The overall objective of this project is to determine the molecular mechanism of activation, signal transduction, and regulation of a prototype G protein coupled receptor (GPCR), i.e., the human muscarinic cholinergic Hm1 receptor. Cholinergic deficits are a hallmark of senile dementias such as Alzheimer, and a fundamental understanding of cholinergic neurotransmission is needed in development of therapeutic strategies. This project employees a genetic approach to determine the functional domains of the Hm1 receptor by extensive mutational analysis, whit focus on three key processes that remain poorly understood for Hm1 specifically and for GPCRs in general. The first includes the molecular mechanisms of receptor activation and signal transduction via G proteins and second messenger pathways. Second, as a result of activation, the receptors undergo rapid cellular trafficking, i.e., sequestration, internalization, and recycling. The third and slower process involves the destruction of functional receptor (down-regulation_. Rapid receptor desensitization is not detectable for Hm1 in several tissue tested, and it is therefore not studied here. This laboratory has constructed numerous Hm1 mutants that permit ne for the first time to dissect these pathways and define the location of several requisite receptor domains. These include the central portion of the second intracellular loop (i2) which was unexpectedly found to play a major role in G protein activation and internalization, an S/T rich domain in the middle of the i3 loop which regulation internalization and subsequent downregulation, and a domain of the i3 loop which mediates a distinct second pathway of downregulation. As such domains have not been identified for any of the GPCRs, their complete characterization will add significantly to our understanding of GPCR regulation. Similar receptor domains exist in most GPCRs, and the general significance of these novel domains will therefore be determine d by mutational analysis of selected additional receptor genes within this large family. Because the regulatory i3 loop domain includes several serine and threonine residues, the involvement of protein kinase mediated phosphorylation in the internalization process will be tested. These studies will be extended to the analysis of similar domains in Hm2, Hm3, beta-2 adrenoceptor, and the thyrotropin releasing hormone (TRH) receptor to test the general validity of the functional domains for GPCRS. The results from this study will clarify the molecular mechanisms and the functional significance of Hm1 activation and cellular trafficking. The responsible receptor domains will then serve as a tool to isolate the target protein mediating receptor functions. Knowledge of Hm1 receptor regulation will benefit therapy of Alzheimer disease with muscarinic agonist.